In order to evaluate the steric and electronic influences of the heme axial ligands on the vibrational modes of heme c, various ferric and ferrous six-coordinate heme c compounds have been prepared from microperoxidase-8 (MP8) and different extrinsic ligands. In this paper, the absorption and Soret-excited resonance Raman (RR) spectra of imidazole, imidazolate, 1-methylimidazole, and histidine complexes of MP8 are presented. The absorption characteristics of the unligated forms, either aggregated or monomeric, as well as of the ligated forms of MP8(III) and MP8(II) have been determined as a function of pH, the presence of a cationic detergent, and the ligand concentration. Spectrophotometric titrations have shown that MP8(III) and MP8(II) can bind one or two molecules of exogenous ligand, forming monoligated or bisligated complexes. The latter form, observed with large excesses of ligand, results from the displacement of the intrinsic proximal His of MP8 by an exogenous ligand. Several structural marker bands have been detected in the high- and low-frequency regions of RR spectra. The high-frequency RR spectra of the ImH compounds of MP8(III) exhibit a v10 mode sensitive to ligand deprotonation(s). Moreover, the replacement of His by an exogenous ImH in MP8(III) complexes induces the upshift of the v10 mode frequency (1637-1641 cm-1), indicating that the porphyrin skeleton is less distorted when the internal coordination of proximal His to heme is broken. A similar dependence of the out-of-plane porphyrin distortion is suggested for the low-frequency mode v8 (343-347 cm-1). As far as the ferrous compounds are concerned, the mode most sensitive to the ImH deprotonation is v11, which is downshifted from 1539 to 1527 cm-1. Comparisons of the low-frequency regions of the RR spectra of imidazole-type ligated MP8(III) and MP8(II) complexes, as well as observations based on isotopic substitutions of the corresponding 1-methylimidazole complexes (MeIm-->MeIm-d6), allow the assignment of two bands in the 184-197 and 400-409 cm-1 regions to modes involving the symmetric and asymmetric stretches of the axial ligands, respectively. Two other bands in the 343-347 and 359-362 cm-1 regions, sensitive to the mass and/or deprotonation states of the axial ligands, have been tentatively assigned to v(Fe-N(pyrrole)) modes coupled to either a deformation mode of axial bonds or an internal mode of the bound imidazole(s).(ABSTRACT TRUNCATED AT 400 WORDS)
The visible absorption and Soret-excited resonance Raman spectra of ferrous microperoxidase-8 [MP8(II)], an octapeptide containing a heme c, are reported. These spectroscopies indicate that MP8(II), dissolved in aqueous buffered solutions, forms low-spin six-coordinated complexes in the 7-14 pH range. Intermolecular bonding interactions of MP8(II) in water account for this behavior. On the contrary, when the hemopeptide is dispersed in aqueous solutions containing detergent or an alcohol, the spectroscopic data show that the iron atom of MP8(II) is essentially high-spin five-coordinated in accordance with a monomeric structure of MP8(II). In addition to a high-spin signature to the heme skeletal modes, the high-frequency regions of resonance Raman spectra characterize an electronic influence of the thioether bridges on the frequency of stretching modes of C beta-C beta bonds (nu 2, nu 11, and nu 29). On the other hand, the low-frequency Raman spectra of monomeric MP8(II) at pH 7.5 present significant differences in the 150-250-cm-1 regions depending upon the solvent composition (pH, presence or absence of detergent, alcohol). These effects are attributed to frequency variations of the Fe-N(His)-involving mode which indicate changes in the H-bonding interactions of the axial His and therefore solvent-dependent changes of the octapeptide conformation. Our resonance Raman data further show that the axial His of monomeric MP8(II) could be totally deprotonated in aqueous cetyltrimethylammonium bromide solution at very alkaline pH (pKa = 13.3). The vibrational data (100-1700 cm-1) obtained for the various monomeric forms of MP8(II) are expected to be useful for determining the heme structure and environment in reduced c'-type cytochromes. Comparisons of resonance Raman data with X-ray crystallographic data available for different hemoproteins allow us to evaluate the ionization and H-bonding states of the His bound to the high-spin five-coordinated hemes. These data are discussed in terms of proximal influence of protein-His-heme interactions on the determination and the regulation of a particular biological function.
Soret-excited resonance Raman (RR) spectra of oxidized and reduced cytochromes ć from Rhodospirillum molischianum and Rhodobacter sphaeroides, in solution, are reported. The spectra of the type I ferricytochromes ć in both species contain different extents of two forms. One of these is readily assignable to a "normal" five-coordinated high-spin heme. The second species with v3 and v10 modes at 1502 and 1635 cm-1, respectively, is attributed to a five-coordinated intermediate-spin heme. The RR data show that the equilibrium between these two forms is species-dependent at neutral pH and 20 degrees C. The v(Fe-His) mode of the a form of reduced cytochromes ć is assigned to a band at 228-231 cm-1, indicating that the proximal His has a strong electronegative character. X-ray crystallographic data on R. molischianum ferricyt ć show that the proximal His has no interaction with either the protein or water molecules [Finzel, B.C., Weber, P.C., Hardman, K.D., & Salemme, F.R.(1985) J. Mol. Biol. 186, 627-643]. Considering that the absence of H bonding at the coordinated histidine corresponds to a low frequency for the v(Fe-His) mode (195-205 cm-1), the structure and/or environment of the proximal histidine appears different for cyt ć (III) in the crystal and cyt ć (II) in aqueous solution. To account for the elevated frequency of the v(Fe-His) mode of cyt ć (II), several possibilities have been examined. Among these, we propose that a conserved Lys residue, located in the protein sequence three residues before the His ligand, can form an electrostatic interaction with the (His)N1 atom, directly or through a water molecule. It is further suggested that this electrostatic interaction could also play a role in the high-spin <--> intermediate-spin equilibrium of oxidized cytochromes ć.
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